Reactive oxygen species (ROS) play an important role in aging. Oxidation of DNA, proteins, lipids, and small molecules by ROS inhibit the actions of these biological components. Protein oxidation by ROS during the aging process is largely attributed to the inactivation and the degradation of proteins. Carbonylation is one mode of protein oxidation that is important in the aging process. It occurs in response to iron-catalyzed, hydrogen peroxide (H2O2)-dependent oxidation of amino acid side chains. Protein carbonylation has been thought to inactivate protein functions and to mark the damaged proteins for proteasome-dependent degradation. While carbonylated proteins are believed not to undergo the electron reduction, my laboratory discovered the protein de-carbonylation mechanism, in which carbonyl groups can be eliminated through the electron reduction. Further, our preliminary experiments identified that glutaredoxin-1 (Grx1) plays a catalytic role in protein de-carbonylation. Major amino acid residues that are susceptible to iron-catalyzed oxidation include proline and arginine, both of which get oxidized to become glutamyl semialdehyde that contains a carbonyl group. Proline residues are oxidized to 5-hydroxyproline that is further oxidized to glutamyl semialdehyde. While the oxidation of 5-hydroxyproline to glutamyl semialdehyde is readily reversible, whether the oxidation of proline to 5-hydroxyproline occurs is unclear. However, based on our recent results on protein de-carbonylation, I hypothesize that the reaction for the oxidation of proline residues to glutamyl semialdehyde is fully reversible through the catalysis by reducing enzymes such as Grx1. Consequently, I also hypothesize that glutamyl semialdehyde that is produced from arginine can also be converted to proline. Further, glutamyl semialdehyde can be oxidized to glutamic acid. This suggests a revolutionizing concept that iron-catalyzed oxidation can convert arginine to proline, arginine to glutamic acid, or proline to glutamic acid within the protein structure, resulting in the occurrence of naturally occurring site-directed mutagenesis. I hypothesize that these modifications result in altered protein functions and contribute to aging. The objective of this R03 project is to provide evidence for the occurrence of arginine-proline, arginine-glutamic acid, and proline-glutamic acid conversions within the protein structure, as novel naturally occurring site-directed mutagenesis processes. The objective of the application will be accomplished by pursuing three specific aims: 1) Define the reduction of 5- hydroxyproline to proline within the protein structure 2) Identify the protein modification that is consistent with the arginine-proline conversion in aging; and 3) Identify protein modifications that are consistent with arginine-glutamic acid or proline-glutamic acid conversion in aging. The proposed work is highly innovative, as it will address for the first time a biologic mechanism that involves naturally occurring site-directed mutagenesis and provide a novel mechanism of ROS actions. Results will be significant because they are expected to provide a new mechanism of aging and help developing strategies to delay the aging process in humans.